Riboswitch Regulation of Aminoglycoside Resistance Acetyl and Adenyl Transferases

نویسندگان

  • Xu Jia
  • Jing Zhang
  • Wenxia Sun
  • Weizhi He
  • Hengyi Jiang
  • Dongrong Chen
  • Alastair I.H. Murchie
چکیده

In our recent paper, we reported an aminoglycoside-sensing RNA in the leader RNA of the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD), enzymes that confer resistance to aminoglycoside antibiotics through modification of the drugs. Our study explains a well-known phenomenon: the induction of expression levels of the AAC/AAD proteins on addition of aminoglycosides (Williams and Northrop, 1976). In this paper, we presented data that showed: (1) aminoglycoside-specific induction of reporter genes mediated by the leader RNA of the AAC in Pseudomonas fluorescens; (2) aminoglycoside binding to the leader RNA by surface plasmon resonance spectroscopy (SPR); (3) that binding induces a structural transition in the leader RNA that can be detected by changes in gel electrophoretic mobility and chemical probing; (4) the identification of a specific aminoglycoside-RNA crosslink; (5) confirmation, via mutational analysis, of the main features of the RNA secondary structure and the importance of structural elements within it for drug binding. We show these aspects of our study to be internally consistent and complementary. The six 4,6 deoxystreptamine aminoglycosides that induce the reporter gene bind well to the leader RNA by SPR and cause a structural transition in the RNA, as measured by chemical probing. The three control molecules that do not induce reporter gene expression bind weakly to the leader RNA by SPR and do not cause a structural transition in the RNA by chemical probing. We have reporter assays, SPR, and chemical probing data that are all in clear and good agreement. In our view, these data are consistent with a riboswitch model of regulation for aac/aad genes. In the accompanying Correspondence in this issue ofCell, Roth and Breaker suggest that the binding that we measure reflects spurious electrostatic drug-RNA interactions. However, the correlation between the in vitro binding that we measure by SPR and the specificity of the reporter gene induction argues against this. Accordingly, the aminoglycosides that bind weakly do not induce the reporter gene, whereas aminoglycosides that bind with high affinity induce leaderRNA-mediated reporter gene expression, suggesting that the binding that we measure is biologically relevant. Moreover, in reporter assays in which the leader RNA of aac/aad is replaced by a different leader RNA (cat-86), we no longer observe induction of the reporter gene, suggesting that the interaction between inducing aminoglycosides and the leader RNA of aac/aad is very specific. Additional roles are emerging for aminoglycosides as cellular modulators and subinhibitory doses induce biofilm formation (Hoffman et al., 2005) and the bacterial SOS response (Baharoglu and Mazel, 2011). Roth and Breaker assert that riboswitches typically influence reporter gene expression over a range of 10to 1000fold, but this is only correct for some riboswitches. A number of riboswitches display lower levels of reporter gene expression; for example, the adenine binding add riboswitch (Lemay et al., 2011) shows 3-fold induction and the guanine riboswitch, 6.7-fold repression (Mandal et al., 2003). Riboflavin represses the FMN riboswitch 5-fold (Lee et al., 2009), and the S-adenosyl methioninebinding SMK riboswitch is repressed 2to 3-fold (Smith et al., 2010). Thus, the levels of reporter gene expression measured in solution in our reporter assay are close to previously characterized translational riboswitches and are consistent with the translational riboswitch that we propose. However, we initially measured reporter gene expression by agar diffusion assays—a more sensitive assay system inwhich induction of gene expression can be directly visualized on agar

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عنوان ژورنال:
  • Cell

دوره 153  شماره 

صفحات  -

تاریخ انتشار 2013